Intestinal stem cell aging at single‐cell resolution: Transcriptional perturbations alter cell developmental trajectory reversed by gerotherapeutics

Abstract The intestinal epithelium consists of cells derived from continuously cycling Lgr5hi intestinal stem cells (Lgr5hi ISCs) that mature developmentally in an ordered fashion as the cells progress along the crypt‐luminal axis. Perturbed function of Lgr5hi ISCs with aging is documented, but the consequent impact on overall mucosal homeostasis has not been defined. Using single‐cell RNA sequencing, the progressive maturation of progeny was dissected in the mouse intestine, which revealed that transcriptional reprogramming with aging in Lgr5hi ISCs retarded the maturation of cells in their progression along the crypt‐luminal axis. Importantly, treatment with metformin or rapamycin at a late stage of mouse lifespan reversed the effects of aging on the function of Lgr5hi ISCs and subsequent maturation of progenitors. The effects of metformin and rapamycin overlapped in reversing changes of transcriptional profiles but were also complementary, with metformin more efficient than rapamycin in correcting the developmental trajectory. Therefore, our data identify novel effects of aging on stem cells and the maturation of their daughter cells contributing to the decline of epithelial regeneration and the correction by geroprotectors.


| INTRODUC TI ON
The intestinal mucosa is a prime example of the decline in regenerative capacity, increase in cellular damage and metabolic dysregulation that characterizes aging (Di Giosia et al., 2022). These changes are linked to age-associated intestinal disorders, with aging also a major risk factor for colorectal cancer (CRC) (Nalapareddy et al., 2022). The potential role of perturbations in Lgr5 hi intestinal stem cells (Lgr5 hi ISCs) is of particular interest in understanding aging since these cells renew and maintain the mucosa through their continuous division at the crypt base (Barker et al., 2007). Reduction in Wnt signaling has been reported as a key that represses stem cell functions of Lgr5 hi cells with aging (Nalapareddy et al., 2017;Pentinmikko et al., 2019), but how it links to the developmental progression of progenitor compartments has not been addressed.
Several pharmacologic approaches have been reported to delay age-related manifestations including reversal of stem cell dysfunction and extension of lifespan (Moskalev, 2020;Partridge et al., 2020). Although none are yet approved for ameliorating the effects of aging in humans, metformin and rapamycin have been studied as regulators of cellular metabolic activities that are linked to aging phenotypes Blagosklonny, 2019;Moskalev et al., 2022;Novelle et al., 2016). However, given their pleiotropic impact on metabolism and linked pathways, understanding the complexity of their effects requires more detailed investigation. The proportion of cells in each cluster/cell type was similar across the different age and treatment groups, suggesting that the overall cellular composition of the intestinal epithelium was maintained for >2 years of age in the mouse ( Figure S1c). However, analysis of gene expression demonstrated a wide range of genes altered in expression with age across clusters and lineages (≥1.5-fold change, coupled with P adj <0.01, Figure 1c), suggesting that transcriptomic profiles in the ostensibly same cell types are reprogrammed with age. For most clusters, treatment of the aged mice with metformin or rapamycin reversed altered expression profiles in cells. Restoration of expression profile was most effective in EC8 cells, identified as mature enterocytes, for both drugs, where it had been altered to the greatest extent in the aged mice ( Figure 1c; Table S1).
The Lgr5 gene, encoding the receptor for the key stem cell growth factor R-spondin, is regulated by Wnt signaling, and in the intestine, is most highly expressed in cells at the crypt base (Barker et al., 2007). These Lgr5 hi cells are considered the major canonical stem cells maintaining the structure and function of the intestinal epithelium (Barker et al., 2007). The mean expression of Lgr5 mRNA in the stem cluster from old mice significantly decreased by 33% compared to that in young mice and was restored back to 87% of young mice by metformin and 83% by rapamycin (Figure 2a). We next focused on Lgr5 hi cell signature genes, defined as those genes altered in expression in the immediate Lgr5 lower progeny of Lgr5 hi cells that have left the stem cell niche and no longer function as stem cells (Muñoz et al., 2012). Of the 467 transcripts in this stem cell signature gene set expressed in Lgr5 hi cells from young mice, 71% were expressed at a lower level in older mice ( Figure 2b).
The distributions of expression levels in young vs old mice, and in young vs old mice treated with either rapamycin or metformin, are shown in Figure 2c, with the percentage of genes at different levels of expression relative to young mice in Table 1. Compartments 1 and 2 are those Lgr5 hi stem cell signature genes repressed by >50% (compartment 1) or 20%-50% (compartment 2, Figure 2c). However, for older mice treated with rapamycin or metformin, this pattern was shifted ( Figure 2c; Table 1). Specifically, as shown in Figure 2c with quantification in Table 1, in old mice, 12% of the signature genes were expressed at or less than 50% of their level in young mice (compartment 1), but this decreased to 5% and 4% for the old mice treated with metformin and rapamycin, respectively. Similarly, expression levels for 38% of the signature genes were 20% to 50% lower in old mice (compartment 2), but the percent of signature genes at these lower levels decreased to 30% and 33% for the old rapamycin and metformin-treated mice, respectively. Reflecting this, the number of genes in compartments 3 and 4, in which expression level changes were within 20% of young mice, increased in the old mice treated with rapamycin or metformin ( Figure 2c; Table 1). These shifts in the cumulative distribution of gene expression ratio were analyzed statistically using the Kolmogorov-Smirnov test (Massey Jr., 1951). Comparison between Old/Young vs Old-treatment/ Young for each drug significantly shifted the distribution closer to 1 (p < 0.001 for each drug). However, the comparison between rapamycin and metformin was not significantly different ( Figure 2d).
Thus, the repressed expression of stem cell signature genes in aged Lgr5 hi cells was significantly rescued by treatment with rapamycin or metformin.
Gene set enrichment analysis (GSEA) identified significantly altered pathways in the stem cell cluster (Figure 2e). Oxidative phosphorylation (OXPHOS), a key metabolic pathway in regulating stem cell function (Rodríguez-Colman et al., 2017;Zhang et al., 2022) was upregulated in the stem cell cluster of older mice, and this was reversed by either metformin or rapamycin. Moreover, the linked metabolic pathways of fatty acid metabolism and glycolysis were also upregulated in the stem cluster of old mice but reversed only by metformin treatment. By contrast, Wnt and cell cycle pathways, which are fundamental for stem cell self-renewal and generation of progeny that undergo differentiation, were suppressed in the stem cluster of old mice, while the ribosome pathway, also important for supporting stem cell function, and downregulated as cells mature during their migration along the crypt-villus axis (Mariadason et al., 2005) was upregulated in the stem cells. Thus, although it is established that OXPHOS is necessary for ISC functions (Rodríguez-Colman et al., 2017), the elevation of this and related fatty acid metabolism in older mice may also perturb stem cell functions, emphasizing the importance of maintaining these pathways within a homeostatic range for normal stem cell functions. This was confirmed by investigating the self-renewal capability of stem cells by analysis of BrdU incorporation prior to sacrifice ( Figure S2a). Quantification of BrdU+ nuclei in crypts confirmed that proliferating cells were reduced in old mice by 28% (p < 0.0001; Figure S2b). Metformin or rapamycin treatment increased proliferating cells back to youthful levels, both highly statistically significant ( Figure S2b), confirming that the proliferation of ISCs was suppressed both transcriptionally and functionally, which were rescued by drug treatments.
Single-cell analysis enables investigating the link between the essential and altered pathways in individual cells. This was investigated using the AddModuleScore function, which calculates the activity score for a pathway in individual cells by accessing gene expression level compared to random control genes (Tirosh et al., 2016). Figure S2c illustrates the correlated distribution of Wnt and cell cycle pathways in individual ISCs (left) and Lgr5 hi ISCs (right) for each condition, the elliptical areas delineating points within 95% of the Gaussian distribution. This shifted towards the lower left with aging but was partially reversed by rapamycin and metformin back towards the informatic space occupied by younger mice. By contrast, coordinate regulation of the ribosome (protein synthesis) and cell cycle pathways showed that the module score was higher for ribosome activity with lower cell cycle activity in aged mice, but the effect of either rapamycin or metformin treatment to shift these back towards the pattern of young mice were less clear ( Figure S2d). Bivariate analysis using MANOVA for two variables based on mean module F I G U R E 1 scRNAseq analysis of small intestinal epithelial cells: (a) Schematic representation of experimental designs; drug treatment was started at age of 21-month-old for 3 months. 3 replicates were used in each group. (b) Cluster map and cell lineages from 12 combined scRNAseq libraries. Abbreviates; R-replicating; R-Div-replicating to dividing; Div-dividing; EC-enterocytes; Sec-secretory progenitor cells; EE-enteroendocrine cells; (c) number of differentially expressed genes (fold change >1.5 & P adj <0.01) in each cluster from the comparison between old vs young (red bar), metformin-treated vs old (green bar), and rapamycin-treated vs old (blue bar)  Differential gene expression analysis  Table 1.
(d) Cumulative density graph of ratio distribution. Statistical analysis was performed using the Kolmogorov-Smirnov test between two groups (***: p < 0.001). (e) Differentially-regulated pathways (P adj <0.05) in the stem cluster using GSEA KEGG pathways; NES-normalized enrichment score. (f) Scatter plot for two variables with mean module score. Mean module scores from each mouse (filled) or each condition (empty, average value) were plotted for Wnt and cell cycle pathways (top) or ribosome and cell cycle pathways (bottom). p value was calculated using MANOVA, assuming each mouse as independent.

| Altered metabolism of ISCs influences their essential functions
With the fact that aging perturbs the self-renewal capability of Lgr5 hi cells, and metabolic changes of ISCs influence how their progeny mature and undergo lineage differentiation (Alonso & Yilmaz, 2018;Beumer & Clevers, 2020;Chandel et al., 2016), we extended analyses to how these cells mature as they progress through progenitor and lineage-specific compartments. Pathway analysis using fold change of differentially expressed genes in old vs young mice showed that at least 79% of the significantly altered pathways in ISCs of old mice continue to be altered in the progenitor cell types sequentially emerging from the stem cell compartment ( Figure S3a). This included the compartments identified as Replicating (R), Replicating-Dividing (R-Div), and Dividing 1 and 2 (Div1, Div2). Therefore, we hypothesized that changes in stem cell expression profile can continue to influence the progressive maturation of cells as they progress through these cell compartments. This was analyzed using Monocle, which predicts the trajectory of cell maturation based on gene expression profiles in individual cells (Trapnell et al., 2014). Assuming cells derived from the Stem cluster (yellow arrow, Figure S3b (Table S2) Table S2). This analysis also revealed that OXPHOS activity differed based on cell position on the trajectory. In the main stem cells, there was a steep difference in mean module score between young and old mice, but the difference did not exist in the side stem cells, implying that aging impaired the balance of metabolic activity determining the function of ISCs (Figure 3e,i). Elevation of OXPHOS in main stem cells from old mice was reversed towards that of young mice with metformin, but rapamycin was not effective for main stem cells, rather affecting side stem cells, reflecting the overall lower efficacy of rapamycin and the distinct mechanisms of action of the two drugs (Figure 3e,i, Table S2). Thus, this rigorous bioinformatic-statistical analysis shows that activities of key cellular functions and metabolic pathways are distinct in relation to position along the intestinal cell developmental trajectory in young mice, compared to old mice. Metformin and rapamycin treatment both augmented key pathway activities.
However, metformin showed a greater effect on adjusting key pathways in main cells, while the rapamycin effect was less dependent on the cellular position along the trajectory.

| Alteration in stem cells delays proper maturation of cells
Analysis of disease pathways for the mainstream cells along the trajectory using Ingenuity Pathway Analysis (IPA) revealed that the stem and progenitor clusters along the trajectory in old mice showed activation of the predefined pathways of organismal death, growth failure, and apoptosis, and complementary inhibition of cell survival pathways, consistent with the potential deteriorating function of the tissue with age (Figure 4a-d). These pathogenic pathways reversed  When all compartments were considered together, the p value was <0.0001, supporting our hypothesis that cell-type transition along the branch points significantly changed with aging.
The relationship between the OXPHOS and cell cycle pathways was investigated along this trajectory using a module score for each pathway as in the earlier analyses. In young mice, the mean module score for OXPHOS between branch points (Figure 4f) gradually increased and then decreased along with the cell-type transition of the developmental trajectory, and showed a sharper rise at the last 2 stages of the trajectory where mature cells initially appear. However, this pattern was greatly attenuated in Old mice. Metformin restored the pattern towards that of young mice, but rapamycin did not.
Similarly, for the cell cycle pathway (Figure 4g), the mean module score increased as the cells emerged from the stem cell compartment, in parallel to known elevated proliferation in subsequent compartments, collectively termed the transit amplifying cells, and then fell sharply at the end of the trajectory in accordance with the appearance of mature enterocytes. By contrast, there was a progressive increase in the cell cycle module score along the trajectory in Old mice, but the sharp decrease at the end of the trajectory seen for Young mice did not occur, suggesting an incomplete process of maturation until the last branch point on the trajectory. However, both metformin and rapamycin restored this overall pattern in old mice to more closely resemble the temporal pattern at these stages in young mice. In summary, our data show a tight linkage between predicted cell identity and metabolic pathways in cell maturation along the developmental trajectory of intestinal epithelial cells, and that aging perturbs the coordination of how these cells are reprogrammed, which leads to this impaired developmental progression of cells. We further investigated the increased OXPHOS pathway using IHC for Ndufb8 of complex I and Cox5b of complex IV of the mitochondrial electron transport chain. Quantitative analysis showed that both Ndufb8 and Cox5b were significantly elevated in the crypts of old mice compared to young mice, consistent with scRNAseq analysis ( Figure S4a-d). Interestingly, neither metformin nor rapamycin reversed these changes at the protein level. The discrepancy in the effect of the drugs between the level of RNA expression of nuclear genes encoding these mitochondrial proteins and its protein level may be due to the fact that mitochondria are long-lived, and that 3 months of treatment with the drugs is not sufficient to alter their structure, whereas in the old mice, substantial remodeling of mitochondria is accumulated with age.
Analysis of the single-cell data had shown a clear delay of cell maturation along the developmental trajectory with aging and its reversal by geroprotectors. This was followed up by an investigation of two specific genes using immunofluorescence. Uhrf1 and Ccnb1 were assayed since their expression level was tightly associated with cell identity: Uhrf1 is highly expressed in early progenitor cells, but Metabolic pathways are critical for sustaining continuous cell cycling of Lgr5 hi ISCs to maintain the mucosa, and metabolic status is also a key in determining whether stem cells retain self-renewal capacity or differentiate following their replication (Mihaylova et al., 2018;Rodríguez-Colman et al., 2017;Wang et al., 2021). Our data show that aged mice exhibited significant alterations in key metabolic pathways such as OXPHOS, glycolysis, and fatty acid metabolism. Therefore, the programming of metabolic pathways is reconfigured in aging with important consequences for stem and progenitor cell functions and mucosal remodeling. Dichotomized analysis clearly showed that there was heterogeneity even among cells within the same cluster as regards to metabolic pathway profile. DAPI Merged developmental progression, while the effect of rapamycin was less tightly linked to their position on the trajectory. Therefore, tracing cellular maturation using trajectory analysis showed that metformintreated mice restored the profile of developmental progression that characterizes the mucosa of young mice, while the effect of rapamycin was more limited. Trajectory analysis is a novel approach to understand the more subtle effects on cell developmental maturation in aging, and similar trajectory analyses based on transcriptional profiles have also recently been used to show altered cell differentiation along the crypt-luminal axis in human ulcerative colitis (Smillie et al., 2019), in ISCs in which mitochondrial composition has been altered (Ludikhuize et al., 2020), and in mice fed a diet that increases the probability for tumor development (Choi et al., 2022).
Aging is a major underlying risk factor for CRC (Nalapareddy et al., 2022), with clear effects of aging on the intestine reported (Baron & Pisani, 2021;Choi et al., 2018;He et al., 2020), and herein deconvolved by single-cell analyses. Of note, despite a major disruption in stem cell function and development of linages, older wildtype mice do not develop tumors spontaneously when fed control diets. However, when wild-type mice are fed a diet long-term that mimics intake levels of common nutrients linked to those consumed in populations at high risk for colon cancer, they do develop small and large intestinal tumors reflecting the etiology, incidence, frequency, and lag with older age of human sporadic colon cancer (Aslam et al., 2010;Newmark et al., 2001;Newmark et al., 2009;Yang et al., 2008). This emphasizes that long-term dietary patterns and aging are inextricably linked as major risk factors for the de- mice were randomized to either the same control diet or a diet in which either 0.1% metformin (Cayman Chem) or microencapsulated rapamycin (eRapa) at a concentration of 42 ppm (Rapamycinholdings,

Inc.) was incorporated into the formulation (TestDiet). Eudragit was
also included in the control and metformin diets (429 ppm) in order to match the rapamycin formulation. All mice were group housed and fed these diets for 3 months under a 14 L:10D photoperiod at 22°C, remained on these formulations for 3 months, and were subsequently euthanized, and intestinal tissue was harvested at either 5 or 24 months of age, respectively, for analysis. 3 replicates for each group were used. All experimental methods were approved by the IACUC at the Albert Einstein College of Medicine.

| Immunohistochemistry
For BrdU staining, immunostaining was performed as previously described. Briefly, antigen retrieval was performed via a citrate buffer (pH = 6) using a pressure cooker for 10 min. After rehydra-

| Image analysis
All image analyses were done with Fiji software. To quantify BrdU+ nuclei, duodenal epithelium was imaged at 60x magnification.
Automatically set threshold was applied to count BrdU+ nuclei. 15 crypts were measured from 3 individual mice in each group. A number of positive nuclei were divided by the total nuclei in the crypt.
To quantify co-stained cells for Uhrf1 and Ccnb1, the duodenal epithelium was imaged at 60x magnification. Nuclei that were positive for fluorescent signal (above top 4% of threshold) were counted, and the percentage of co-stained cells was calculated over the sum of the number of cells positive for each staining. 15 crypts were analyzed from 3 individual mice in each group. To quantify OXPHOS subunit proteins, duodenal and jejunal crypts were imaged at 20x magnification. Automatically set threshold was applied to measure positive areas for signal. The area above the threshold was then divided by the total area of crypt. Measurement was conducted up to 60 μm from the bottom of the crypts to minimize size variability.
17 crypts from 3 mice were measured. One-way ANOVA followed by the Tukey's multiple comparisons test was applied to calculate p value. Statistical analysis was performed using GraphPad Prism version 0.3.1 for Mac, GraphPad Software, San Diego, California USA, www.graph pad.com. treated as random effects. The computation is performed by R function glmer.nb. Two-sample Kolmogorov-Smirnov test was used to compare two distributions (in Figure 2d). Multivariate analysis of variance was used to compare mean module scores at two pathways among the four experimental groups (in Figure 2f) using R function MANOVA assuming each mouse was independent.

| Statistical analysis
Figure 3b-i was tested using one-way ANOVA followed by the Tukey's multiple comparison test (Table S2), which was performed using GraphPad Prism version 0.3.1 for Mac, GraphPad Software, San Diego, California USA, www.graph pad.com. Figure 4e was tested using Pearson's Chi-squared test with a simulated P value.
Individual comparison of each compartment treated each mouse as independent. Figure 5c was tested using one-way ANOVA followed by the Tukey's multiple comparison test.

ACK N O WLE D G E M ENTS
Supported in part by P30 AG038072, 5T32AG023475-20 from the NIA, and R01CA214625, R01CA229216, R01CA222358, and P30-013330 from the NCI.

CO N FLI C T O F I NTE R E S T S TATE M E NT
All authors state that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T scRNAseq data sets have been deposited in the Gene Expression
Omnibus (GEO) database under the accession code: GSE210669.